Approach 1: Understand and improve on the protective efficacy of ALVAC &gp120 vaccination. In the last several years we have investigated vaccine approaches for HIV, based on DNA alone or in combination with poxvirus-based vaccines, and found that these vaccine modalities are able to elicit mainly memory T-cells, and when combined with gp120 protein boost can induce high titer binding antibodies. A similar strategy using the canary pox vector ALVAC in combination with gp120 was used in the recently completed RV144 trial in Thailand that showed marginal efficacy (31.2%) [Jim Tartalia NejM 2009]. The relative efficacy of similar vaccine modalities varied in animal models dependent on co-receptor usage of the virus used in challenge experiments, the viral strain, the age of the animals, and the route and dose of exposure. Recent evidence suggest that the dose of challenge exposure to SIVmac251, a CCR5 user virus, affects the read out of vaccine efficacy and that at high doses of challenge exposure, vaccine protection is diminished and several virus variants are transmitted. It is estimated that in humans, the risk of HIV transmission ranges by different routes between ten and one thousand per encounter and that when transmission occurs, only few HIV variants pass the mucosa. We explored the relevance of the SIVmac251 macaque model to vaccine efficacy in humans by using vaccines similar to those used in the AIDSVAX or the RV144 trials, and by using a dose of SIVmac251 intended to transmit few viral variants to better mimic HIV mucosal transmission in humans. We found that, in these conditions, the animal model faithfully recapitulated the efficacy of these vaccine modalities in humans. Now that we have defined an appropriate model that recapitulates the protection we are designing studies aimed at defining the correlates of protection induced by ALVAC and gp120. In attempt to improve upon the immunogenicity of ALVAC and gp120 and to investigate the importance of challenge dose on evaluating vaccine efficacy. We evaluated the effect of varying the SIVmac251 challenge dose on vaccine efficacy after immunizing with combination of a DNA, ALVAC-SIV, and gp120. The evaluation of vaccine efficacy in the non-human primate model for HIV has been often carried out using a single high dose challenge modality. While it is now clear that low repeated doses mucosal exposure to SIV/SHIV better recapitulate sexual transmission of HIV-1 in humans, it is still not known whether a relationship exists between viral inoculum size and vaccine efficacy. Thus, we investigated the impact of the mucosal challenge dose on the efficacy of DNA-SIV/ALVAC-SIV/ gp120 protein, a strategy similar to the Thai trial vaccine. For the first time, we have demonstrated that vaccine efficacy depends on the dose of challenge. In fact while vaccination transiently decreased viral replication after a single intra-rectal dose of 6100 TCID50, it protected 3 of the 12 macaques challenged with repeated low doses of 470 TCID50 SIVmac251. Vaccination also resulted in prolonged protection from high viral replication, reduction of the number of transmitted variants and systemic and mucosal CD4+ T cell regeneration in the animals exposed to 470 TCID50, but not to 6100 TCID50 SIVmac251. Interestingly, while the temporary protection observed in the high dose challenged group was correlated with CD8 T cell responses only, both antibodies and CD8 cell responses to gag and env participated to the prolonged protection from viral replication. Our data demonstrate the importance of accuratly modeling HIV transmission in macaques then evaluating of vaccine efficacy, with particular attention to the dose of exposure. Approach 2: Targeting the Vaginal Mucosa with Human Papillomavirus Pseudovirion Vaccines delivering SIV DNA The majority of HIV infections occur via sexual transmission. HIV must survive the genital tract, cross the vaginal or rectal epithelium and then disseminate throughout the body. The failure of current vaccine strategies at providing sterilizing immunity may be due to the quantity, quality or durability of vaccine induced responses at the portal of entry. Blocking vaginal transmission of HIV may require vaccines that target the female genital tract and induce local immunity. HIV vaccines based on viral vectors, proteins, or a combination thereof, tested in phase III vaccine efficacy trials in humans, induced mainly systemic immune responses using vaccines delivered by intramuscular inoculation. While these vaccine modalities induce variable levels of HIV-specific responses in the blood, little is known about their ability to induce mucosal responses. A subset of human papillomavirus (HPVs) are sexually transmitted mucosal pathogens that naturally infect cervico-vaginal keratinocytes. HPV-VLP-based vaccines are safe and very effective at preventing the HPV infections that cause cervical neoplasia in women. HPV capsid proteins, L1 and L2, can self assemble into virus like particles (VLPs) and, when co-transfected with a plasmid containing a gene of interest, L1 and L2 will encapsidate the plasmid forming pseudovirions (PsVs). Human Papilloma Virus (HPV) pseudovirions, is a technology developed by my collaborators, Drs Buck, Schiller, and Lowy, at the NCI. HPV PsVs have been shown to effectively deliver reporter genes to the female genital tract in multiple animal models. HPV PsVs infection is limited to keratinocytes and requires minor disruption of the epithelium. Expression of the transgene is robust but transient, lasting approximately seven days in the mouse genital tract. Furthermore, HPV PsVs may serve as adjuvants, engaging toll like receptors and facilitating the activation and maturation of antigen presenting cells. We have exploited the ability of HPV PsVs to target the female genital tract and used PsVs as vectors to deliver DNA encoding SIV genes to a site of SIV transmission in two non-human primate species. We demonstrated that HPV PsVs, used as vehicles for the delivery of SIV DNA, induced SIVmac251 specific cellular and humoral immune responses at the genital tract, in the genital draining lymph nodes, and in blood in both cynomolgus and rhesus macaques. Intravaginal vaccination results in the recruitment of both CD4+ and CD8+ T-cells to the site of vaccination, increasing the absolute number of T-cells in the cervico-vaginal mucosa. In addition, HPV PsVs induced mucosal immune responses that rapidly expanded upon vaginal exposure to SIVmac251. We are currently investingating if HPV PsV induced immune responses can protect macaques from a repeated low dose vaginal challenge with SIVmac251. Approach 3: Induce effector T-cells in the gastrointestinal tract using heat shock gp96-Igbased SIV recombinant vaccines. This is part of a collaborative effort with Dr Podack from the University of Miami and includes the preparation of cellular SIV-gp96-vaccines secreted by 293 cells. This cell-based gp96-Ig vaccine has prolonged in vivo secretion of gp96-Ig-peptide that imitates viral replication and provide immune stimuli comparable to attenuated viruses. The vaccine is composed of transfected 293 cells that secrete ER-chaperone gp96-Ig in complex with SIV derived peptides. Strong rectal and vaginal CTL reponses are achieved by i.p. immunization of macaques with the gp96-Ig-SIV vaccine. In the past year we have found that coinjection of recombinant gp120 protein with the 293-gp96SIVIg-vaccine cells provides significant protection from rectal SIV infection. As neither gp96-SIV alone or gp120-protein alone provides protection from infection we conclude that both mucosal CTL and antibodies are required to protect against mucosal SIV challenge. In the upcoming years we will asses methods of improving the protective efficacy of gp96+ gp120 vaccination.